• Title/Summary/Keyword: E. coli LacZ Gene

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Detection of Chlorotoluene and Nitrotoluene Compounds by Recombinant Microbial Biosensors (재조합 미생물 바이오센서를 이용한 chlorotoluene과 nitrotoluene 화합물의 검출)

  • Lee, Da Young;Cho, Jae Ho;Lim, Woon Ki;Shin, Hae Ja
    • Journal of Life Science
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    • v.24 no.1
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    • pp.54-60
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    • 2014
  • Aromatic hydrocarbons are toxic environmental pollutants that are detrimental to the ecosystem and human health. Among them, chlorotoluene and nitrotoluene are toxic to hydrobios and irritate the skin, eyes, and respiratory organs of humans. We herein report the development of recombinant microbial biosensors for cheap and rapid monitoring of chlorotoluene and nitrotoluene compounds. Plasmids were constructed by inserting the xylR regulatory gene for BTEX (benzene, toluene, ethylbenzene, and xylene) degradation into upstream of Po' (the DmpR activator promoter Po with the deletion of its own upstream activating sequences) or Pu (the cognate promoter of XylR)::lacZ (the ${\beta}$-galactosidase gene) and transformed into Escherichia coli $DH5{\alpha}$. In the presence of inducers, the biosensor cells immobilized in agarose developed a red color in 1-2 h due to the hydrolysis of chlorophenol red ${\beta}$-D-galactopyranoside (CPRG), a substrate of ${\beta}$-galactosidase that was expressed by the inducers. Among BTEX, high responses were specifically observed with o-, m-, p-chlorotoluene ($0.1{\mu}M-100 mM$) and o-, m-, p-nitrotoluene (0.1 mM-100 mM). Po' demonstrated higher responses than those with Pu. The biosensors immobilized in agarose showed good stability after 21 days' storage at $4^{\circ}C$, and responses in untreated wastewater spiked with chlorotoluene and nitrotoluene, suggesting they can be used to detect compounds in wastewater.

Retrovirus Vector-Mediated Inductional Expression of the Human Lactadherin Gene in Mouse Mammary Epithelial Cells (Mouse Mammary Epithelial Cell에서 Retrovirus Vector를 이용한 Human Lactadherin 유전자의 유도적 발현)

  • 권모선;구본철;정병현;염행철;박창식;김태완
    • Korean Journal of Animal Reproduction
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    • v.27 no.1
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    • pp.15-23
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    • 2003
  • Lactadherin (formerly known as BA46), a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and breast carcinomas and may prevent symptomatic rotavirus infections. In this study, under the control of tissue specific and hormonal inducible mouse whey acidic protein (WAP) promote., the expression pattern of lactadherin (Ltd) in lactogenic hormone-dependent mouse mammary epithelial cell line HC11 were tested. pLNWLtd construct containing 2.4 kilobases of the WAP promoter and 1.5 kilobases of human lactadherin gene was stably transfered into HC11 cells using retroviral vector system. Integration and expression level of the transgene was estimated using PCR and RT-PCR, respectively. Prominent induction of Ltd gene under the WAP promoter was accomplished in the presence of insulin, hydrocortisone and prolactin. Compared to the control (cells cultured with insulin alone), however we observed that the WAP promoter was leaky. These data indicate that luther studies are needed in finding an appropriate promoter other than WAP promoter because of its leakiness.

Tumor Surpressor Gene Therany, and Natural Product with Vectors[Aoenouirus, Aoenn associated virus] in Human Papilloma virus (HPV[Human papilloma virus]유래 바이러스 벡터[Adenovirus, Adeno associated virus]를 이용한 암 억제유전자치료법과 자연산물에서의 암 억제 효과)

  • 천병수;노민석;유종수;김준명
    • KSBB Journal
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    • v.16 no.6
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    • pp.579-591
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    • 2001
  • The cell growth inhibitor effect of cervical cancer cells was investigated by liposome mediated transfection (pRcCMVp53/lipofectin) and by transfection using adenovirus (AdCMVp57). The papilloma virus cancer cell lines we used in this study were HPV16 positive, having inhibiter gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3. LacZ gene of E.coli was used as the marker gene for the transfection efficiency. The effect on the inhibition of tumor cell growth was measured by cell count and cell viability though ELISA analysis and MTT assay. The inhibition of tumor cell growth was confirmed by measuring each assay for six days, comparing with the normal control cell growth. The cell growth of cervical cancer calls by transfection was significantly reduced and showed tittle differences among the cell lines. To eliminate the potential problem of Ad(adenovirus) contamination during rAAV production, rAAV can be produced by a triple transfection of vector plasmic, packaging plasmid, and adenovirus helper plasmid. To examine the helper functions of Ad plasmids on the production of rAAV vector, we carried out cotransfection of three plasmids, AAV vector, packaging construct, and Ad helper plasmids. The optimized transfection condition for calcium phosphate method is 25ug of total DNA per 10-cm-diameter plate of 293 cell. We found that rAAV yields peaked at 48hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/ml based on the quantification of viral DNA. Recent1y, Kombucha(fungi) was identified as a very potent antileukefic agent. In the present study, effect of natural toxin(plankton) and Kombucha is PSP(GTXI-3, neoSTX), on various MTT assay cervical cancer cell line. Toxin(GTX 1-3, neoSTX) also inhibited the proliferation in primary cervical cancer calls in a dose-dependent toxin concentration. These results showed that toxin was very potent in inhibiting the proliferation of cervical cancer calls in vitro. Toxins and Kombuoha exhibited a dose dependent inhibition of cellular proliferation in cancer cell line.

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Development of pSJE6c, an Expression Vector for Kimchi Lactic Acid Bacteria, and Heterologous Gene Expression Using the Vector (김치유산균용 발현벡터 pSJE6c 개발과 이를 이용한 외래 유전자 발현)

  • Lee, Kang-Wook;Park, Ji-Yeong;Lee, Ji-Yeon;Lee, Hwang-A;Baek, Chang-Un;Jo, Hyeon-Deok;Kim, Joo-Yeon;Kwon, Gun-Hee;Chun, Ji_Yeon;Kim, Jeong-Hwan
    • Microbiology and Biotechnology Letters
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    • v.37 no.4
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    • pp.389-398
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    • 2009
  • Development of expression vectors is important for the basic and applied researches on kimchi LAB (lactic acid bacteria). An expression vector, pSJE6c was constructed by inserting P6C promoter sequence from Lactococcus lactis into pSJE, a shuttle vector for E. coli and Leuconostoc species. To test the efficiency of pSJE6c, aga ($\alpha$-galactosidase) and lacZ ($\beta$-galactosidase) genes were expressed in Lactobacillus brevis 2.14. Compared to the pSJE, expression levels of both genes were increased, indicating P6C promoter was better than indigenous promoters. Enzyme activities of L. brevis cells harboring pSJE6caga (pSJE6c with aga) or pSJE6Z (pSJE6c with lacZ) were 1.5-2 fold higher than those with pSJEaga (pSJE with aga) or pSJEZ (pSJE with lacZ). More RNA transcripts were detected in cells harboring pSJE6c based recombinant plasmid. The results indicated that heterologous gene expressions in kimchi LAB could be improved significantly by use of efficient expression vectors.

vfr, A Global Regulatory Gene, is Required for Pyrrolnitrin but not for Phenazine-1-carboxylic Acid Biosynthesis in Pseudomonas chlororaphis G05

  • Wu, Xia;Chi, Xiaoyan;Wang, Yanhua;Zhang, Kailu;Kai, Le;He, Qiuning;Tang, Jinxiu;Wang, Kewen;Sun, Longshuo;Hao, Xiuying;Xie, Weihai;Ge, Yihe
    • The Plant Pathology Journal
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    • v.35 no.4
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    • pp.351-361
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    • 2019
  • In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant $G05{\Delta}phz{\Delta}prn::lacZ$ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant $G05{\Delta}vfr$ and $G05{\Delta}phz{\Delta}prn::lacZ{\Delta}vfr$. By quantifying ${\beta}-galactosidase$ activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant $G05{\Delta}vfr$, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.

Functional Expression of an Anti-GFP Camel Heavy Chain Antibody Fused to Streptavidin (Streptavidin이 융합된 GFP항원 특이적인 VHH 항체의 기능적 발현)

  • Han, Seung Hee;Kim, Jin-Kyoo
    • Journal of Life Science
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    • v.28 no.12
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    • pp.1416-1423
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    • 2018
  • With strong biotin binding affinity ($K_D=10^{-14}M$), the tetrameric feature of streptavidin could be used to increase the antigen binding activity of a camel heavy chain (VHH) antibody through their fusion, here stained with biotinylated horseradish peroxidase and subsequent immunoassays ELISA and Western blot analysis. For this application, we cloned the streptavidin gene amplified from the Streptomyces avidinii chromosome by PCR, and this was fused to the gene of the 8B9 VHH antibody which is specific to green fluorescent protein (GFP) antigens. To express a soluble fusion protein in Escherichia coli, we used the pUC119 plasmid-based expression system which uses the lacZ promoter for induction by IPTG, the pelB leader sequence at the N-terminus for secretion into the periplasmic space, and six polyhistidine tags at the C-terminus for purification of the expressed proteins using an $Ni^+$-NTA-agarose column. Although streptavidin is toxic to E. coli because of its strong biotin binding property, this soluble fusion protein was expressed successfully. In SDS-PAGE, the size of the purified fusion protein was 122.4 kDa in its native condition and 30.6 kDa once denatured by boiling, suggesting the tetramerization of the monomeric subunit by non-covalent association through the streptavidin moiety fusing to the 8B9 VHH antibody. In addition, this fusion protein showed biotin binding activity similar to streptavidin as well as GFP antigen binding activity through both ELISA and Western blot analysis. In conclusion, the protein resulting from the fusion of an 8B9 VHH antibody with streptavidin was successfully expressed and purified as a soluble tetramer in E. coli; it showed both biotin and GFP antigen binding activity suggesting the possible production of a tetrameric and bifunctional VHH antibody.

Optimization of Retrovirus Mediated-Gene Transfer into Hematopoietic Stem Cells (Retrovirus를 이용하여 조혈모세포에 유전자를 전달하기 위한 최적화)

  • Kim, Sang-Gyeong;Seo, Heon-Seok;Lee, Jong-Won;Sin, Dong-Geon;Lee, Jae-Gwan;Kim, Hyeon-Min;Kim, Jae-Sik;Seo, Jang-Su
    • KSBB Journal
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    • v.14 no.5
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    • pp.593-599
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    • 1999
  • In this study, optimal conditions to infect CD34 positive cells containing hematopoietic stem cells obtained from cord blood and bone marrow were found using two different retroviral vectors expressing human growth hormone (hGH) and $\beta$-galactosidase. CD34 positive cells were successfully infected with recombinant retroviruses only when the CD34 positive cells were co-cultured with packaging cells secreting recombinant retroviruses. To find the highest infection efficiency for the gene transfer, CD34 positive cells from cord blood were co-cultured with packaging cells secreting recombinant retroviruses encoding E. coli lacZ gene. The highest infection efficiency was obtained when CD34 positive cells were cultured for 3 days, and then co-culturing was done for another 2 days. When CD34 positive cells from bone marrow were co-cultured with packaging cells secreting recombinant retroviruses encoding hGH gene, the maximum amount of hGH was also secreted at the same conditions found above, i.e. 3 days of culture and 2 days of co-culture. These results show that there are optimal conditions for the gene transfer into hematopoietic stem cells regardless of sources of target cells or retroviral vectors used to infect.

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Isolation and characterization of sigH from Corynebacterium glutamicum (Corynebacterium glutamicum의 sigH 유전자의 분리 및 기능분석)

  • Kim Tae-Hyun;Kim Hyung-Joon;Park Joon-Sung;Kim Younhee;Lee Heung-Shick
    • Korean Journal of Microbiology
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    • v.41 no.2
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    • pp.99-104
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    • 2005
  • Corynebacterial clones which exert regulatory effects on the expression of the glyoxylate bypass genes were isolated using a reporter plasmid carrying the enteric lacZ fused to the aceB promoter of Corynebacterium glutamicum. Some clones carried common fragments as turned out by DNA mapping technique. Subcloning analysis followed by the measurement of $\beta-galactosidase$ activity in Escherichia coli identified the region responsible for the aceB-repressing activity. Sequence analysis of the DNA fragment identified two independent ORFs of ORF1 and ORF2. Among them, ORF2 was turned out to be responsible for the aceB-repressing activity. ORF1 encoded a 23,216 Da protein composed of 206 amino acids. Sequence similarity search indicated that the ORF may encode a ECF-type $\sigma$ factor and designated sigH. To identify the function of sigH, C. glutamicum sigH mutant was constructed by gene disruption technique and the sigH mutant showed growth retardation as compared to the wild type strain. In addition, the mutant strain showed sensitivity to oxidative-stress generating agent plumbagin. This result imply that sigH is probably involved in the stress response occurring during normal cell growth.

CONSTRUCTION OF HNGF-$\beta$ RECOMBINANT ADENOVIRUS & SCREENING OF ITS EXPRESSION AFTER TRANSFECTION INTO VARIOUS CELL LINES (말초신경재생을 위한 hNGF-$\beta$ recombinant Adenovirus의 제작 및 수종세포주에서 신경성장인자의 발현)

  • Gao, En-Feng;Chung, Hun-Jong;Ahn, Kang-Min;Kim, Yoon-Tae;Park, Hee-Jung;Sung, Mi-Ae;Kim, Nam-Yeol;Yoo, Sang-Bae;Myoung, Hoon;Hwang, Soon-Jung;Kim, Myung-Jin;Kim, Sung-Min;Jang, Jeong-Won;Lee, Jong-Ho
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.27 no.5
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    • pp.446-456
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    • 2005
  • Nerve growth factor(NGF) has a critical role in peripheral nerve regeneration. The aim of this study is to construct a well-functioning hNGF-$\beta$ recombinat adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with adenovirus mediated hNGF-$\beta$ gene transfection into Schwann cells. First PCR associated cloning of GFP-tagged hNGF-$\beta$ which was ligated into E1/E3 deleted adenoviral vector was performed and tranfected into E. coli to construct hNGF-$\beta$ recombinant adenovirus. After production of recombinat adenovirus in a large scale, its transfection efficiency, expression, and function were evaluated using cell lines or primarily cultured cells of HEK293 cells, Schwann cells, fibroblast(NIH3T3) and myocyte(CRH cells). GFP expression was observed in 90% of infected cells compared to uninfected cells. Total mRNA isolated from hNGF-$\beta$ recombinat adenoviru infected cells showed strong RT-PCR band, however, LacZ recombinant adenovirus infected or uninfected cells did not. NGF quantification by ELISA showed a maximal release of 18.865 +/- 0.31ng/mL at 4th day. PC-12 cells exposed to media with hNGF-$\beta$ recombinant adenovirus infected Schwann cell demonstrated higher levels of differentiation compared with controls. We generated hNGF-$\beta$ recombinant adenovirus and induced over expression of NGF successfully in nonneuronal and neuronal cells. Following these result, it is expected to develop an improved treatment strategy peripheral nerve regeneration using the hNGF-$\beta$ gene transfected cells.